The present invention relates to kappa3-related-opioid receptor-3 (KOR-3) splice variant polypeptides, to KOR-3 splice variant polynucleotides, to methods of screening compositions for agonists and antagonists of the splice variant receptor activities and to methods of measuring splice variant polypeptide binding activities.
Opiates are drugs derived from opium and include morphine, codeine and a wide variety of semisynthetic opioid congeners derived from them and from thebaine, another component of opium. Opioids include the opiates and all agonists and antagonists with morphine-like activity and naturally occurring endogenous and synthetic opioid peptides. Morphine and other morphine-like opioid agonists are commonly used pharmaceutically to produce analgesia.
There are now many compounds with pharmacological properties similar to those produced by morphine, but none has proven to be clinically superior in relieving pain. References to morphine herein will be understood to include morphine-like agonists as well. The effects of morphine on human beings are relatively diverse and include analgesia, drowsiness, changes in mood, respiratory depression, decreased gastrointestinal motility, nausea, vomiting, and alterations of the endocrine and autonomic nervous systems. Pasternak (1993) Clin. Neuropharmacol. 16:1. Doses of morphine need to be tailored based on individual sensitivity to the drug and the pain-sparing needs of the individual. For instance, the typical initial dose of morphine (10 mg/70 kg) relieves post-operative pain satisfactorily in only two-thirds of patients. Likewise, responses of an individual patient may vary dramatically with different morphine-like drugs and patients may have side effects with one such drug and not another. For example, it is known that some patients who are unable to tolerate morphine may have no problems with an equianalgesic dose of methadone. The mechanisms underlying variations in individual responses to morphine and morphine-like agonists have not been defined.
The analgesic effects of morphine are transduced through opioid receptors in the central nervous system (CNS), located at both spinal and multiple supraspinal sites. Morphine and other agonists induce profound analgesia when administered intrathecally or instilled locally into the dorsal horn of the spinal cord. Several mechanisms of action are believed to mediate the inhibition of nociceptive reflexes from reaching higher centers of the brain, including the inhibition of neurotransmitter release by opioid receptors on the termini of primary afferent nerves and post synaptic inhibitory actions on interneurons and on the out-put neurons of the spinothalamic tract.
Profound analgesia can also be produced by the instillation of morphine into the third ventricle or within various sites in the midbrain and medulla, most notably the periaqueductal gray matter, the nucleus raphe magnus, and the locus ceruleus. Although the neuronal circuitry responsible has not been defined, these actions produce enhanced activity in the descending aminergic bulbospinal pathways that exert inhibitory effects on the processing of nociceptive information in the spinal cord. Simultaneous administration of morphine at both spinal and supraspinal sites results in a synergized analgesic response, with a ten-fold reduction in the total dose of morphine necessary to produce equivalent analgesia at either site alone.
Morphine also exerts effects on the neuroendocrine system. Morphine acts in the hypothalamus to inhibit the release of gonadotropin releasing hormone (GnRH) and corticotropin-releasing factor (CRF), thus decreasing circulating concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), and adrenocorticotropin (ACTH), and xcex2-endorphin. As a result of the decreased concentrations of pituitary trophic hormones, the concentrations of testosterone and cortisol in the plasma decline. The administration of opiates increases the concentration of prolactin (PRL) in plasma, most likely by reducing the dopaminergic inhibition of PRL secretion. With chronic administration, tolerance eventually develops to the effects of morphine on hypothalamic releasing factors.
Opiates can interfere with normal gastrointestinal functioning. Morphine decreases both gastric motility and the secretion of hydrochloric acid in the stomach. Morphine may delay passage of gastric contents through the duodenum for as long as 12 hours. Morphine also decreases biliary, pancreatic, and intestinal secretions and delays the digestion of food in the small intestine. Propulsive peristaltic waves in the colon are diminished or abolished after administration of morphine and commonly, constipation occurs. For a detailed review of the physiologic effects of morphine, see Reisine and Pasternak (1996) Goodman and Gilman""s The pharmacological basis of therapeutics, Ninth Edition (Hardman et al. eds.) McGraw-Hill pp 521-555.
Morphine also exerts effects on the immune system. The most firmly established effect of morphine is its ability to inhibit the formation of rosettes by human lymphocytes. The administration of morphine to animals causes suppression of the cytotoxic activity of natural killer cells and enhances the growth of implanted tumors. These effects appear to be mediated by actions within the CNS. By contrast, xcex2-endorphin enhances the cytotoxic activity of human monocytes in vitro and increases the recruitment of precursor cells into the killer cell population; this peptide also can exert a potent chemotactic effect on these cells. A novel type of receptor (designated xcex5) may be involved. These effects, combined with the synthesis of proopiomelanocortin (POMC) and preproenkephalin by various cells of the immune system, have stimulated studies of the potential role of opioids in the regulation of immune function. Sibinga and Goldstein (1988) Annu. Rev. Immunol. 6:219.
Side effects resulting from the use of morphine range from mild to life-threatening. Morphine causes constriction of the pupil by an excitatory action on the parasympathetic nerve innervating the pupil. Morphine depresses the cough reflex through inhibitory effects on the cough centers in the medulla. Nausea and vomiting occur in some individuals through direct stimulation of the chemoreceptor trigger zone for emesis, in the postrema of the medulla. Therapeutic doses of morphine also result in peripheral vasodilatation, reduced peripheral resistance and an inhibition of baroreceptor reflexes in the cardiovascular system. Additionally, morphine provokes the release of histamines, which can cause hypotension. Morphine depresses respiration, at least in part by direct effects on the brainstem regulatory systems. In humans, death from morphine poisoning is nearly always due to respiratory arrest. Opioid antagonists can produce a dramatic reversal of severe respiratory depression and naloxone is currently the treatment of choice. High doses of morphine and related opioids can produce convulsions, which are not always relieved by anticonvulsant agents, such as naloxone.
The development of tolerance and physical dependence with repeated use is a characteristic feature of all opiates. Dependence seems to be closely related to tolerance, since treatments that block tolerance to morphine also block dependence. In vivo studies in animal models demonstrate the importance of neurotransmitters and their interactions with opioid pathways in the development of tolerance to morphine. Blockade of glutamate actions by noncompetitive and competitive NMDA (N-methyl-D-aspartate) antagonists blocks morphine tolerance. Trujillo and Akil (1991) Science 251:85; and Elliott et al. (1994) Pain 56:69. Blockade of the glycine regulatory site on NMDA receptors has similar effects to block tolerance. Kolesnikov et al. (1994) Life Sci. 55:1393. Administering inhibitors of nitric oxide synthase in morphine-tolerant animals reverses tolerance, despite continued opioid administration. Kolesnikov et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:5162. These studies indicate several important aspects of tolerance and dependence. First, the selective actions of drugs on tolerance and dependence demonstrate that analgesia can be dissociated from these two unwanted actions. Second, the reversal of preexisting tolerance by NMDA antagonists and nitric oxide synthase inhibitors indicates that tolerance is a balance between activation of processes and reversal of those processes. These observations suggest that, by use of selective agonists or antagonists, tolerance and dependence in the clinical management of pain can be minimized or disassociated from the therapeutic effects.
In addition to morphine, there are a variety of opioids suitable for clinical use. These include, but are not limited to, Levorphanol, Meperidine, Fentanyl, Methadone, Codeine, Propoxyphene and various opioid peptides. Certain opioids are mixed agonists/antagonists and partial agonists. These include pentazocine, nalbuphine, butorphanol, and buprenorphine. The pharmacological effects of levorphanol closely parallel those of morphine although clinical reports suggest that levorphanol produces less nausea.
Meperidine exerts its chief pharmacological effects on the CNS and the neural elements in the bowel. Meperidine produces a pattern of effects similar but not identical to those described for morphine. In equianalgesic doses, meperidine produces as much sedation, respiratory depression, and euphoria as morphine. The pattern of unwanted side effects that follow the use of meperidine are similar to those observed after equianalgesic doses of morphine, except that constipation and urinary retention are less common.
Fentanyl is a synthetic opioid estimated to be 80 times as potent as morphine as an analgesic. High doses of fentanyl can result in severe toxicity and produce side effects including muscular rigidity and respiratory depression.
Methadone is an opioid with pharmacological properties similar to morphine. The properties of methadone include effective analgesic activity, efficacy by the oral route and persistent effects with repeated administration. Side effects include detection of miotic and respiratory-depressant effects for more than 24 hours after a single dose, and marked sedation is seen in some patients. Effects on cough, bowel motility, biliary tone and the secretion of pituitary hormones are qualitatively similar to those of morphine. In contrast to morphine, codeine is approximately 60% as effective orally as parenterally, both as an analgesic and as a respiratory depressant.
Codeine has an exceptionally low affinity for opioid receptors, and the analgesic effect of codeine is due to its conversion to morphine. However, codeine""s antitussive actions probably involve distinct receptors that bind codeine specifically.
Propoxyphene produces analgesia and other CNS effects that are similar to those seen with morphine. It is likely that at equianalgesic doses the incidence of side effects such as nausea, anorexia, constipation, abdominal pain, and drowsiness would be similar to those of codeine.
Opioid antagonists have therapeutic utility in the treatment of overdosage with opioids. As understanding of the role of endogenous opioid systems in pathophysiologic states increases, additional therapeutic indications for these antagonists will emerge. If endogenous opioid systems have not been activated, the pharmacological actions of opioid antagonists depend on whether or not an opioid agonist has been administered previously, the pharmacological profile of that opioid and the degree to which physical dependence on an opioid has developed. The antagonist naloxone produces no discernible subjective effects aside from slight drowsiness. Naltrexone functions similarly, but with higher oral efficacy and a longer duration of action. Currently, naloxone and naltrexone are used clinically to treat opioid overdoses. Their potential utility in the treatment of shock, stroke, spinal cord and brain trauma, and other disorders that may involve mobilization of endogenous opioids remains to be established.
The complex interactions of morphine and drugs with mixed agonist/antagonist properties are mediated by multiple classes of opioid receptors. Opioid receptors comprise a family of cell surface proteins, which control a range of biological responses, including pain perception, modulation of affective behavior and motor control, autonomic nervous system regulation and neuroendocrinological function. There are three major classes of opioid receptors in the CNS, designated mu, kappa and delta, which differ in their affinity for various opioid ligands and in their cellular distribution. The different classes of opioid receptors are believed to serve different physiologic functions. Olson et al. (1989) Peptides 10:1253; Lutz and Pfister (1992) J. Receptor Res. 12:267; and Simon (1991) Medicinal Res. Rev. 11:357. Morphine produces analgesia primarily through the mu-opioid receptor. However, among the opioid receptors, there is substantial overlap of function as well as of cellular distribution.
Members of each known class of opioid receptor have been cloned from human cDNA and their predicted amino acid sequences have been determined. Yasuda et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6736; and Chen et al. (1993) Mol. Pharmacol. 44:8. The opioid receptors belong to a class of transmembrane spanning receptors known as G-protein coupled receptors. G-proteins consist of three tightly associated subunits, alpha, beta and gamma (1:1:1) in order of decreasing mass. Following agonist binding to the receptor, a conformational change is transmitted to the G-protein, which causes the G-alpha subunit to exchange a bound GDP for GTP and to dissociate from the beta and gamma subunits. The GTP-bound form of the alpha subunit is typically the effector-modulating moiety. Signal amplification results from the ability of a single receptor to activate many G-protein molecules, and from the stimulation by G-alpha-GTP of many catalytic cycles of the effector.
Most opioid receptor-mediated functions appear to be mediated through G-protein interactions. Standifer and Pasternak (1997) Cell Signal. 9:237. Antisense oligodeoxynucleotides directed against various G-protein alpha subunits were shown to differentially block the analgesic actions of the mu-, delta-, and kappa-opioid-agonists in mice. Standifer et al. (1996) Mol. Pharmacol. 50:293.
The amino acid sequences of the opioid receptors are approximately 65% identical, and they have little sequence similarity to other G-protein-coupled receptors except for somatostatin. Reisine and Bell (1993) Trends Neurosci. 16:506. The regions of highest similarity in sequence are the sequences predicted to lie in the seven transmembrane-spanning regions and the intracellular loops. Regions of amino acid sequence divergence are the amino and carboxy termini and the second and third extracellular loops.
Each receptor subtype has a characteristic pattern of expression. Mu-opioid receptor mRNA is present in the periaqueductal gray, spinal trigeminal nucleus, cuneate and gracile nuclei, and thalamus regions of the brain involved in pain perception and associated with morphine analgesia (Defts et al. (1994) J. Comp. Neurol. 345:46); in nuclei involved in control of respiration, consistent with the ability of morphine to depress respiration; and in neurons of the area postrema, where morphine has been shown to cause nausea and induce vomiting. Other consequences of mu-opioid receptor activation include miosis, reduced gastrointestinal motility, and feelings of well-being or euphoria. Pasternak (1993). The pattern of mu-opioid receptor mRNA expression correlates with the brain centers involved in mediating the biological actions of morphine and mu-selective agonists. Delta-opioid receptor mRNA is found in the dorsal horn of the spinal cord. Kappa1-opioid receptor mRNA is expressed in the hypothalamic regions, which may account for many of the neuroendocrine effects of the kappa selective agonists.
Alternative splicing has been observed with a number of G-protein-coupled receptors, including somatostatin 2 (Vanetti et al. (1998) FEBS Lett. 311:290), dopamine D2 (Guiramand et al. (1995) J. Biol. Chem. 270:7354), prostaglandin EP3 (Namba et al. (1993) Trends Pharmacol. Sci. 16:246), serotonin receptor subtypes 5-HT4 and 5-HT7 (Lucas and Hen. (1995) Trends Pharmacol. Sci. 16:246) and KOR-3. Bare et al. (1994) FEBS Lett. 354:213; and Zimprich et al. (1995) FEBS Lett. 359:142.
Several opioid receptor splice variants have been identified and characterized. At least two MOR-1 splice variants are known, the human MOR-1A and the rat MOR-1BI Bare et al. (1994); and Zimprich et al. (1995). The hMOR-1A splice variant consists of exons 1, 2, 3 and a new exon 3a, and was determined to possess ligand binding characteristics similar to the full-length MOR-1. Bare et al. (1994).
In the case of the Kappa-opioid receptor, few variants have been found. This member of the opioid receptor family was cloned from human (ORL-1; hereinafter hKOR-3), mouse (KOR-3; hereinafter mKOR-3) and other species. U.S. Pat. No. 5,747,279; Bunzow et al. (1994) FEBS Lett. 347:284-288; Fukuda et al. (1994) FEBS Lett. 343:42-46; Wick et al. (1994) Molec. Brain Res. 27:37-44; Pan et al. (1996) Gene 171:255-260; Mollereau et al. (1994) FEBS Lett. 341:33-38; and Pan et al. (1995) Mol. Pharmacol. 47:1180-1188.
The structure of mouse KOR-3 gene has been defined as having five exons separated by four introns. Pan et al. (1996). Although structurally homologous with the cloned traditional opioid receptors, mKOR-3 has low affinity for most opioids and opioid peptides. The ligand for KOR-3 has been identified and designated orphanin FQ/nociceptin (OFQ/N). Reinscheid et al. (1995) Science 270:792-794; and Meunier et al. (1995) Nature 377:532-535. OFQ/N is intimately involved with pain perception, but its actions are complex. Initially, it was reported to be hyperalgesic and that low doses reverse the actions of opioids. Reinscheid et al. (1995); Meunier et al. (1995); Mogil et al. (1996) Neurosci. Lett. 214:1-4; Mogil et al. (1996) Neurosci. 75:333-337; Grisel et al. (1996) NeuroReport 7:2125-2129; Tian et al. (1997) Pharmacol. 120:676-680; Zhu et al. (1997) Neurosci. Lett. 235:37-40; and King et al. (1998) Biochem. Pharmacol. 55:1537-1540. Yet OFQ/N is also an analgesic. Tian (1997); Rossi et al. (1997) J. Pharmacol. Exp. Ther. 282:858-865; Rossi et al. (1996) Eur. J. Pharmacol. 31:R7-R8; King et al. (1997) Neurosci. Lett. 223:113-116; and Yamamoto et al. (1997) Neurosci. 81:249-254.
The complex pharmacology of OFQ/N and antisense studies of KOR-3 raised the possibility of multiple OFQ/N receptors. Rossi et al. (1997); Rossi et al. (1996); King et al. (1997); Mathis et al. (1997) Biochem. Biophys. Res. Commun. 230:462-465; and Pan et al. (1995). Radiolabeled OFQ/N binding to brain homogenates is quite distinct from that to the cloned receptor and is consistent with binding site heterogeneity in the brain. Mathis et al. (1997); Reinscheid et al. (1995); and Pan et al. (1996) FEBS Lett. 395:207-210. Antisense mapping of the three coding exons of the receptor encoded by KOR-3 also raised the question of alternative splicing. Pan et al. (1994) Regul. Pept. 54:217-218; Pan et al. (1995); Rossi et al. (1997); Rossi et al. (1997); and Pasternak et al. (1995) Trends Pharmacol. Sci. 16:344-350. Antisense probes targeting the first coding exon blocked OFQ/N hyperalgesia, but not analgesia, whole other probes targeting the second and third exons blocked analgesia and not hyperalgesia. The second and third coding exons, but not the first, also have been implicated in kappa3 analgesia. These observations raised the possibility that the kappa3 receptor and KOR-3 might result from alternative splicing of the same gene. Pan et al. (1996).
Two alternative splice KOR variants have been reported, including a rat variant (XOR1L) which contains a 28 amino acid residue insertion between 15 base deletion corresponding to Tyr71-Arg75 in the first intracellular loop. Wang et al. (1994) FEBS Lett 1994 348:75-79; and Halford et al. (1995) J. Neuroimmunol. 59:91-101.
Availability of polynucleotide sequences encoding opioid receptor splice variants, and the corresponding polypeptide sequences, will significantly increase the capability to design pharmaceutical compositions, such as analgesics, with enhanced specificity of function. In general, the availability of these polypeptide sequences will enable efficient screening of candidate compositions. The principle in operation through the screening process is straightforward: natural agonists and antagonists bind to cell-surface receptors and channels to produce physiologic effects; certain other molecules can produce physiologic effects and act as therapeutic pharmaceutical agents. Thus, the ability of candidate drugs to bind to opioid receptor splice variants can function as an extremely effective screening criterion for the selection of pharmaceutical compositions with a desired functional efficacy and specificity.
The invention encompasses KOR-3 splice variant polypeptides.
The invention further encompasses a KOR-3 splice variant polynucleotide, including those encoding KOR-3 splice variant polypeptides.
The invention further encompasses methods of screening compositions for an opioid activity by obtaining a control cell that does not express a recombinant or endogenous opioid receptor, obtaining a test cell that expresses a recombinant KOR-3 splice variant polypeptide, contacting the control cell and test cell with an amount of an opioid sufficient to exert a physiologic effect, separately measuring the physiologic effect of the composition on the control cell and test cell and comparing the physiologic effect of the composition to the physiologic effect of the opioid, where determination of a physiologic effect of the composition is expressed relative to that of the opioid.
The invention further encompasses methods of screening compositions for an opioid activity by obtaining a control polypeptide that is not a recombinant opioid receptor, obtaining a test polypeptide that is a recombinant KOR-3 splice variant polypeptide, contacting a composition with the control polypeptide and the test polypeptide, contacting the test polypeptide with an amount of an opioid sufficient to measurably bind the test polypeptide, measuring the binding of the composition and the opioid, and comparing the test polypeptide binding of the composition to that of the opioid, where determination of binding of the composition is expressed relative to that of the opioid.
The invention further encompasses methods of screening compositions for differential opioid activity comprising obtaining a first and second test polypeptide that are KOR-3 splice variant polypeptides and contacting each with a composition, measuring the binding affinity of the composition to the first and second test polypeptides and comparing the binding of the composition and the first test polypeptide to that of the second test polypeptide where differential activity is expressed as a ratio of the two binding affinities.
The invention further encompasses a non-human animal in which one or both endogenous KOR-3 alleles has been altered by homologous recombination with an exogenously introduced nucleic acid provided herein.
The invention further encompasses a non-human transgenic animal carrying a transgene comprising a KOR-3 splice variant polynucleotide.
The invention further encompasses a method for regulating morphine analgesia in a subject by altering the amount of KOR-3 polypeptide activity. Activity can be regulated by administering antigen binding fragments, agonists, antagonists or small molecule ligands to a subject in an amount of and a duration sufficient to regulate morphine analgesia. The antigen binding fragment, agonist, antagonist or small molecule ligand is directed to an KOR-3 splice variant polypeptide.
Opioid activity can also be regulated by administering a DNA plasmid vector containing a KOR-3 splice variant polynucleotide. The DNA plasmid vector thereby expresses an KOR-3 polynucleotide in a subject in an amount of and a duration sufficient to regulate morphine analgesia. Activity can also be regulated by administering an antisense nucleic acid complementary to a KOR-3 splice variant polynucleotide, thereby blocking gene expression in a subject in an amount of and a duration sufficient to regulate morphine analgesia.
The invention further encompasses a method for regulating body weight in a subject by altering the amount of KOR-3 polypeptide activity. Activity can be regulated by administering antigen binding fragments, agonists, antagonists or small molecule ligands to a subject in an amount of and a duration sufficient to regulate body weight. The antigen binding fragment, agonist, antagonist or small molecule ligand is directed to an KOR-3 splice variant polypeptide.
Activity can also be regulated by administering a DNA plasmid vector containing a KOR-3 splice variant polynucleotide. The DNA plasmid vector thereby expresses an KOR-3 polynucleotide in a subject in an amount of and a duration sufficient to regulate body weight. Activity can also be regulated by administering an antisense nucleic acid complementary to a KOR-3 splice variant polynucleotide, thereby blocking gene expression in a subject in an amount of and a duration sufficient to regulate body weight.
The invention further encompasses a method for diagnosing an KOR-3 splice variant-associated pharmacological abnormality, comprising measuring the amount of variant activity or tissue distribution thereof in a subject and comparing that activity or tissue distribution to a control sample, wherein a difference in the amount of activity or tissue distribution correlates with the presence of a pharmacological defect.
The invention further encompasses a method for diagnosing an KOR-3 splice variant-associated disorder of the neuroendocrine system, comprising measuring the amount of variant activity or tissue distribution thereof in a subject and comparing that activity or tissue distribution to a control sample, wherein a difference in the amount of activity or tissue distribution correlates with the presence of a disorder of the neuroendocrine system.
The invention further encompasses antigen-binding fragments specific for the KOR-3 splice variants described herein.